Table Of Content7
Jundishapur J Nat Pharm Prod.2012; (1):17-21.
Jundishapur Journal of Natural
Pharmaceutical Products
www.jjnpp.com
An In Vivo Toxicological Study Upon Shallomin, the Active Antimicrobial
Allium hirtifolium
Constitute of Persian Shallot ( , Boiss) Extract
Mansor Amin 1, Mohammad Hassan Pipelzadeh 2*, Manijeh Mehdinejad 1, Iran Rashidi 3
1 Department of Microbiology, School of Medicine, Infection and Tropical Diseases Research center, Ahvaz Jundishapur University of Medical Sciences,
Ahvaz, IR Iran
2 Department of Pharmacology and Toxicology Research Centre, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, IR Iran
3 Department of Pathology, School of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, IR Iran
A R T I C L E I N F O A B S T R A C T
Article type: Background:
Previous studies have shown that shallomin, one of the active constituents
Original Article of Persian shallot, has a broad range of antimicrobial properties.
Objectives:
The safety of shallomin must be established before it can be used in clinical
Article history:
applications. Therefore, the aim of the present study was to evaluate the acute toxic ef-
Received: 10 Oct 2011 fects of shallomin and to estimate its lethal dose low (LDLo) value.
Materials and Methods:
Revised: 20 Nov 2011 Two series of experiments were performed: In the first series,
Accepted: 10 Dec 2011 we used functional testing to assess the acute toxic effects of shallomin on the blood,
liver, and kidney and examined histopathological changes in the liver, kidney, lung, and
Keywords:
heart, following 7 days of daily intraperitoneal administration of 3 standard doses (10,
Shallots 20, and 30 µg/g body weight of mice). In the second series, the LDLo value was estimated
Acute Toxicity by determining daily mortality in mice after 7-day administration of escalating doses of
shallomin (10 to 240 µg/g body weight of mice).
Results:
The results showed that shallomin (at the anticipated in vivo doses), unlike the
placebo (ethanol), did not produce any adverse effects on the tested organs. The LDLo
value was observed to be 160 µg/g body weight; this value is 8- to 32-times the anticipated
in vivo dose that produces antimicrobial effects under in vitro conditions against vari-
ous pathogenic organisms.
Conclusions:
In conclusion, the results of the present study show that shallomin is a
relatively safe agent, although its use needs to be carefully monitored. Further in vivo
chronic toxicity tests need to be performed to establish the therapeutic potential of shal-
lomin as an antimicrobial agent.
Copyright c2012 DocS.
Implication for health policy/practice/research/medical education:
If found safe, this novel natural product may be a useful alternative in treatment of infections.
Please cite this paper as:
Amin M, Pipelzadeh MH, Mehdinejad M, Rashidi I. An In Vivo Toxicological Study Upon Shallomin, the Active Antimicrobial Consti-
Allium hirtifolium Jundishapur J Nat Pharm Prod 7
tute of Persian Shallot ( , Boiss) Extract. . 2012; (1):17-21.
1. Background Allium
, which includes onion and garlic, and grows wild
Alliumhirtifolium in the Zagros Mountains, Iran. Previous in vitro studies
The Persian shallot ( ), sometimes mis-
Alliumascalonium have shown that the crude extract of the bulb of this
takenly named , belongs to the genus
plant has antimicrobial properties against a variety of
Pseudomonas aeruginosa
Corresponding author: pathogenic bacteria, such as
* Mohammad Hassan Pipelzadeh, Department of
Pharmacology and Toxicology Research Centre, Ahvaz Jundishapur Uni- (1). In vivo investigations showed that 40 mg/mL of the
versity of Medical Sciences, Ahvaz, IR Iran. Tel.: +98-6114458849, Fax: +98- topically applied aqueous extract was useful in the recov-
E-mail:
6114458849, [email protected] ery of rats from burn injuries (2). After autoclaving, the
crude aqueous extract of shallot, unlike onion and garlic,
Copyright c2012 DocS.
18 Amin M et al. An In Vivo Acute Toxicological Study on Shallomin on Blood, Kindey and Liver
2. Objectives
maintained its antimicrobial activity against both gram-
Staphy-
negative and gram-positive pathogenic bacteria (
lococcusaureus Listeriamonocytogenes Bacilluscereus A. The aim of this study was to evaluate the toxicity of
, , ,
fumicans Serratia marcescens Escherichia coli P. aerugi- shallomin on various body systems from 3 prospective
, , ,
nosa Camp. Salmonella typhi S. paratyphi viewpoints: first, by using a range of doses that correlate
, species (3), , A,
Proteus mirabilis Shigella with effective in vitro concentrations to assess its effect
, and species), with a minimum
on biochemical parameters related to the blood, liver,
inhibitory concentration (MIC) ranging from 5 to 20 µg/
and kidney; second, to assess the acute histopathological
mL (2). In addition, the crude extract showed fungistatic
Micros- changes in various organs; and third, to estimate the le-
and fungicidal activity against pathogenic fungi (
porum gypseum Aureobasidium pullulans Trichophyton thal dose low (LDLo) value, which is the lowest reported
, ,
mentagrophyte T. rubrum Fusarium oxysporum Saccharo- concentration that causes death in humans or animals
, , ,
myces cerevisiae Aspergillus niger A. flavus A. fumigatus (4). These types of evaluations are believed to be essen-
, , , ,
Candida albicans tial when collecting preliminary information on the
and ), with a MIC range of 0.15 to 20 mg
possible use of this novel agent in clinical practice. This
µg/mL (2). The active compound responsible for the an-
evaluation was thought to be essential in establishing
timicrobial properties is a flavonoid with the general
preliminary information on the possible use of shallo-
formula C HO and the proposed name of shallomin
14 8 8 min in clinical practice.
(3). Therefore, this ancient plant is a potential source for
the treatment of bacterial and fungal infections and has 3. Materials and Methods
a unique property not commonly found in current anti-
infective agents. However, evidence of the safety of any
3.1. Extraction of Shallomin
medicinal agent, including those obtained from plants,
is required before it can be used in the food industry or
About 300 g of white shallot bulbs (collected in the
in any clinical setting.
spring from the Zagros Mountains; 50 km from Dezful,
Table 1.
Mean ± SEM SD values of various experimental parameters measured after 1 week of daily administration of low (10 µg/g), intermediate (20
µg/g), and high dose (30 µg/g) of shallomin in the shallomin-treated groups, in comparison with that in the ethanol- (placebo) and non-treated groups.
C C C C C C C C C C C C
PLT HCT HGB RBC WBC ALK ALT AST DBil TBil Cr BUN
Low dose 10 µg/g 849.4 ± 54.8 40.6 ± 0.69 13.5 ± 0.22 7a ± 0.1 8.6a ± 2.8 515 ± 47 2.66 ± 0.3 1.6 ± 0.11 0.27 ± 0.01 0.61 ± 0.01 0.51 ± 0.03 81 8 ± 0.9
Intermediate 835.6 ± 30 42.4 ± 1.45 13.3 ± 0.43 7.2 ± 0.2 9.9 ± 0.7 482 ± 29 3.3 ± 0.5 1.4 ± 0.26 0.26 ± 0.01 0.56 ± 0.02 0.5 ± 0.03 8 ± 0.8
dose 20 µg/g
High dose 30 856.5 ± 29 38.1 ± 0.7 12.7 ± 0.24 6.9 ± 0.2 9.6 ± 0.8 424 ± 29 2.2 ± 0.5 1.4 ± 0.42 0.24 ± 0.01 0.61 ± 0.02 0.46 ± 0.03 10 ± 0.7
µg/g
Placebo- ethanol 560.6 ± 48 37.7b ± 0.23 12.1b ± 0.21 7.04 ± 0.21 9.44 ± 0.35 466 ± 29 3 ± 0.16 1.1 ± 0.21 0.23 ± 0.01 0.48b± 0.02 0.55b ± 0.03 9.3b± 0.35
Negative control 765.9a ± 43 45.7a ± 1.1 15.4b ± 0.3 8a ± 0.2 7.3 ± 0.8 462 ± 43 6.9 a ± 0.4 1.55 ± 0.2 0.27 ± 0.01 0.77a ± 0.02 0.82a ± 0.04 8.7 ± 0.6
a P < 0.001, compared to the placebo group (placebo effect).
b P < 0.01, compared to the shallomin-treated groups.
c Abbreviations: ALK: Alkaline Phosphate; BUN: Blood Urea Nitrogen; Cr: Creatinine; DBil: Direct bilirubin; HGB: Hemoglobin; PLT: Platelet; PT: Prothrom-
bin Time; RBC: Red Blood Cells; TBil: Total Bilirubin; WBC: White Blood Cells.
Table 2.
Cumulative number of deceased mice per group, following 1 week of daily intraperitoneal administration of escalating doses (10 to 240 µg/g
body weight) of shallomin dissolved in 0.1 mL ethanol. The lowest dose that caused mortality was observed with 160 µg body weight on the day following
shallomin administration of shallomin administration.
Group-Dose/Body Weight, µg/g Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7
0 0 0 0 0 0 0 0
10 0 0 0 0 0 0 0
20 0 0 0 0 0 0 0
40 0 0 0 0 0 0 0
60 0 0 0 0 0 0 0
80 0 0 0 0 0 0 0
100 0 0 0 0 0 0 0
120 0 0 0 0 0 0 0
160 0 2 3 3 3 3 3
200 0 3 4 5 6 7 6
240 0 5 6 6 6 7 7
Jundishapur J Nat Pharm Prod. 2012;7(1):17-21
An In Vivo Acute Toxicological Study on Shallomin on Blood, Kindey and Liver Amin M et al. 19
a city in Southern Iran) was washed thoroughly in water 80, 120, 160, 200, or 240 µg/g body weight). The mortality
and cut into small pieces by using a kitchen mixer. The rate in each group was assessed daily for 1 week, and the
pieces were then soaked in 300 mL of distilled water and lowest dose that produced mortality during this period
stirred using a magnetic stirrer for 5 h. Then, the suspen- was recorded.
sion was filtered through Whatman No. 1 filter paper,
3.5. Statistical Analysis
and the aqueous extract was mixed with ethyl acetate in
a 50:50 ratio and stirred for 10 min. The upper organic
The results were expressed as mean ± standard error
layer was separated using a separating funnel and centri-
values. The significance of the differences between the
fuged at 5,000 rpm for 10 min. The ethyl acetate layer was
mean values was assessed using analysis of variance, fol-
then removed and transferred into a clean flask. This pro-
lowed by Duncan’s test for multiple comparisons among
cess was repeated 3 times, and the extracts were pooled P
the different groups. A value <0.05 was considered sig-
and dried in a rota-evaporator (Heidolph, Germany) at
nificant.
50°C. The yield from the extract was weighed and dis-
solved in ethanol at various concentrations. 4. Results
3.2. Animal Grouping CBC analysis of the ethanol-treated group showed a
significant reduction in platelet and red blood cell (RBC)
A total of 16 groups (n = 10 per group) of male BALB/c
counts, in comparison with those in both the non-treat-
mice (body weight, 20 ± 5 g) were used. The groups were
ed control and shallomin-treated groups; this suggested
housed in separate cages maintained at room tempera-
that ethanol causes significantly acute suppression of
ture, ranging from 25°C to 30°C, with free access to stan-
the bone marrow. Shallomin, at the doses used, reversed
dard laboratory food and tap water. The mice were used
the platelet-reducing effect of ethanol. However, the
in 2 separate series of experiments: in the first series of in
ethanol-treated group showed significant reduction in
vivo experiments, 5 groups (n = 10 per group) were used
hematocrit and serum hemoglobin levels, which were
for the assessment of hematotoxicological, hepatotoxi- Table
partially reversed in the shallomin-treated group (
cological, and renal toxicological effects with 3 increas- 1
). Furthermore, the white blood cell (WBC) counts in-
ing anticipated doses for the treatment of infections. In
creased in the ethanol-treated group; however, shallo-
the second series designed for assessment of acute toxic-
min had no effect on the ethanol-induced increase in the
ity, 11 groups of healthy male mice were used. Table 1
WBC count ( ).
3.3. Evaluation of Hematotoxicity, Hepatotoxicity, and With respect to hepatic toxicity, ethanol alone caused
significant reduction in ALT, AST, and total bilirubin lev-
Renal Toxicity Effects of Shallomin
els. These changes were not observed following shallo-
In this series of experiments, the first 3 groups (n = 10 min treatment; however, the total serum bilirubin level
per group) were used as the test groups, in which shal- was within the normal range after shallomin treatment.
lomin was administered to the mice at varying doses of However, the direct bilirubin level (conjugated form of
10, 20, or 30 µg/g body weight. The fourth group was used bilirubin) in the ethanol- and shallomin-treated groups
as the placebo group, in which 0.1 mL of ethanol (the did not differ from that in the non-treated group. The
solvent) was administered daily via intraperitoneal (IP) serum creatinine level decreased significantly in the eth-
injection for 7 consecutive days. The fifth group was used anol-treated group, but did not change in the shallomin-
as the non-treated group. treated groups, in comparison with that in the control
Parameters such as complete blood count (CBC), serum group. In addition, the serum BUN level increased in
alkaline phosphatase, aspartate aminotranferase (AST), the ethanol-treated group, but it was within the normal
Table 1
alanine transferase (ALT), creatinine, total bilirubin, di- range in the shallomin-treated group ( ).
rect bilirubin, and blood urea nitrogen (BUN) were mea-
4.1. Histopathological Findings after Shallomin Admin-
sured after 7 days. In addition, the mice were killed, and
istration
their lung, liver, kidney, and heart were removed and
evaluated for any pathological changes. The dose range
No remarkable histopathological changes were ob-
for measurement of these parameters ranged from 5 µg/
served in the lung, liver, heart, or kidneys of the different
mL to 20 µg/mL (adjusted to body weight in g) and was
treatment groups.
selected on the basis of previous in vitro concentrations
observed to be effective against both fungi and bacteria 4.2. Lethality of Shallomin in Mice
(2).
After 7 days of daily escalating doses of shallomin, the
3.4. Estimation of Lethal Dose Low of Shallomin in Mice percentage of mice that died was found to proportional-
Table 2
ly increase in a dose-dependent manner ( ). Among
Seven groups of mice (n = 10 per group; average weight,
the selected groups, the first incidence of death was ob-
20 ± 5 g) were administered IP injection of shallomin dis-
served on the second day of administration with a dose
solved in 0.1 mL of 96% ethanol daily for 7 days (10, 40, Table 2
of 160 µg of shallomin per gram of body weight ( ).
Jundishapur J Nat Pharm Prod. 2012;7(1):17-21
20 Amin M et al. An In Vivo Acute Toxicological Study on Shallomin on Blood, Kindey and Liver
5. Discussion
that, at the doses tested, both ethanol and shallomin had
no adverse effects on hepatic function. In fact, ALT levels,
Assessment of toxicity of an agent, regardless of its
a marker for acute toxicity in hepatic cells, had decreased
source, is an integral part for its future use as a thera-
in both groups.
peutic agent. Previous studies (1, 5-7) had evaluated the
The doses used in this study were selected on the basis
antibacterial properties of raw shallot extract; however,
of concentrations found effective under in vitro condi-
none of the studies had attempted to isolate and test
tions against most pathogenic fungi and bacteria. Pre-
the active ingredient responsible for this property. To
vious in vitro studies showed that the aqueous extract
complement our previous in vitro studies on shallomin,
of crude shallot had both antibacterial and antifungal
we evaluated the toxic effects of shallomin (the active
effects, comparable to conventionally used antibiotics,
antimicrobial constituent of shallot) in mice. One of the
with an antifungal MIC range of 0.15 to 20 mgµg/mL and
main limitations of our present study was the solubility
an antibacterial MIC range of 5 to 20 mgµg/mL (2). Fur-
of shallomin, which is soluble in ether, methanol, and
thermore, in vivo studies using a burn model found an
ethanol. We selected ethanol because it is considered the
effective topical dose of 4 mg/mL. These concentrations
safest solvent. However, acute biochemical toxicity was
are within the normal range of activity of most antibiot-
observed in this group; this highlighted the significant
ics. However, the LDLo value of shallomin was found to
toxic effects of this agent. With respect to the assessed
be 160 µg per gram of body weight; this value, in terms
parameters, variable results in the ethanol-treated
of the ratio of lethality dose to effective concentration,
group primarily affected the blood, kidney, and hepatic
is between 8- and 32-times the in vivo dose anticipated
systems of the mice. In addition, the number of platelets
to produce antimicrobial effects against various patho-
decreased in the ethanol-treated group, but not in the
genic organisms. These findings suggest that shallomin
shallomin-treated groups; this finding suggested that
is a relatively safe agent; however, the in vivo and clinical
shallomin has a protective effect against the platelet-
uses of shallomin need to be carefully monitored. Fur-
reducing effects of ethanol. These toxic effects seem to
ther chronic toxicity tests and clinical experiments need
be related to the bone marrow suppression property of
to be performed to establish the therapeutic potential of
ethanol in acute doses. The nature of such suppressive ef-
shallomin as an antimicrobial agent.
fects on the bone marrow needs to be assessed further in
future studies. In addition, ethanol significantly reduced Acknowledgments
the RBC count and reduced the hemoglobin and hema-
tocrit levels. The shallomin-treated groups showed simi- We thankfully acknowledge the Deputy of Research Af-
lar effects, suggesting that these observed effects on the fairs at the Ahvaz Jundishapur University of Medical Sci-
hemopoietic system were mainly caused by ethanol and ences for the financial support for this project.
that shallomin had no direct toxic effects on this system.
Financial Disclosure
Previous studies reported isolation of various constitu-
ents identified as flavonoids, saponins, and lectins from
None declared.
shallomin (8, 9). However, none of these compounds pos-
sessed antibacterial properties. Ascalin, a unique specific Funding/Support
anti-fungal agent without antibacterial activity (10), was
isolated and identified by Wang and Nag in 2001. This As stated in the acknowledgments financial support of
peptide, derived from another species of shallot (A. as- this project was provided by the Deputy of Research Af-
calonicum), has a molecular weight of 9.5 kDa and pos- fairs of AJUMS.
sesses an N-terminal sequence of YQCGQGG.
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